Development of a Microelectrode Array Sensing System for Water Quality Monitoring

Utah State University DigitalCommons@USU

All Graduate Theses and Dissertations Graduate Studies

5-2008

Robert D. Gardner Utah State University

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DEVELOPMENT OF A MICROELECTRODE ARRAY SENSING

SYSTEM FOR WATER QUALITY MONITORING

Robert D. Gardner

A thesis submitted in partial fulfillment Of the requirements for the degree

MASTER OF SCIENCE

Biological Engineering

Approved:

______Dr Anhong Zhou Dr James W. Burns Major Professor Committee Member

______Dr Ronald Sims Dr Byron R. Burnham Committee Member Dean of Graduate Studies

UTAH STATE UNIVERSITY Logan, Utah

2008

iii ABSTRACT

Development of a Microelectrode Array Sensing

System for Water Quality Monitoring

Robert D. Gardner, Master of Science

Utah State University, 2008

Major Professor: Dr. Anhong Zhou Department: Biological and Irrigational Engineering

This thesis reports the design and fabrication of a low‐cost reliable microelectrode

array sensing platform and its application toward water quality monitoring, including heavy

metal ion detection. Individually addressable microelectrodes were designed in a planar

array on a nonconductive glass substrate by a photolithography method. The size, shape,

composition, and functionality of the microelectrodes were theoretically explored in order

to maximize performance.

The microelectrode array sensing platform was proven and characterized in the

K3Fe(CN)6 electrochemical standard using cyclic voltammetry. The sensor platform exhibited well defined voltammograms and had increased sensitivity relative to a

commercially available microelectrode of similar size. Feasibility for application to heavy

metal ions, copper and lead, detection in aqueous solutions was demonstrated utilizing the

electrochemical method of anodic stripping voltammetry. Well defined voltammograms for

the copper and lead ions were obtained with individual microelectrodes of the sensor

platform, and compared against the similar sized commercially available microelectrode;

increased sensitivity was observed. iv Finally, a piecewise proving technique was done to prove the feasibility for future

coupling of the microelectrode array sensor platform with a previously developed

homemade electrochemical device. By analyzing the response of the homemade electrochemical device compared to that of a commercially available electrochemical analyzer, feasibility was demonstrated.

(153 pages)

v ACKNOWLEDGMENTS

I would like to especially thank Dr. Anhong Zhou for being my advisor, mentor, and friend throughout my time working in his lab and on this thesis. I would like to thank my committee members, Dr. Ronald Sims and Dr. Jim Burns, for their valuable assistance, thoughts, and inspiration for completion of this project. I am also grateful the financial supports from Utah Water Research Laboratory (UWRL), National Institute of Health, USU

College of Engineering, and the Dean’s Office Teaching Funds.

I also thank the lab colleagues from Dr. Zhou’s lab for their help during my studies.

I would like to express my gratitude to Brian Baker from the MicroFab Lab at

University of Utah to provide me with great help with the training and use of their microfabrication facilities there.

I am thankful to Dr. Yangquan Chen and his lab, especially Mr. Austin Jenson, who helped a lot with the setup of homemade SWV box.

I would like to especially thank my wonderful family, friends, and colleagues who have supported me throughout the duration of this project; they never tired of helping me.

My strongest gratitude is reserved for Patricia Korey; thank you for being so patient with me.

Robert Gardner vi CONTENTS

Page

ABSTRACT ...... iii

ACKNOWLEDGMENTS ...... v

CONTENTS ...... vi

LIST OF TABLES ...... ix

LIST OF FIGURES ...... x

CHAPTER

1 PROJECT INTRODUCTION ...... 1 1.1 Problem Description ...... 1 1.2 Overview of the Solution ...... 2

1.2.1 Stripping Voltammetry Analysis ...... 3 1.2.2 Square Wave Anodic Stripping Voltammetry (SWASV) and Differential Pulse Anodic Stripping Voltammetry (DPASV) ...... 4 1.2.3 Microelectrode and Nonlinear Diffusion ...... 5 1.2.4 Combination of Voltammetry and Microelectrode ...... 7

1.3 Aims ...... 7 1.4 Outline of Technical Contents ...... 8

2 DESIGN AND CHARACTERIZATION ...... 9

2.1 Voltammetric Criteria for Testing Utilizing Microelectrodes ...... 9 2.2 Experimental ...... 14

2.2.1 Photolithography Fabrication of MEA ...... 14 2.2.2 MEA Sensor Fabrication and Construction ...... 16 2.2.3 Electrochemical Measurements and Reagents ...... 18

2.3 Results and Discussion ...... 20

2.3.1 Comparison of CV Results of MEA Microelectrode and 2 mm Au Electrode ...... 20 2.3.2 Repeatability of CV Results for Single MEA Electrode ...... 22 2.3.3 Repeatability of 18 MEA Electrodes in 0.3 mM and 0.5 mM K3Fe(CN)6 Solutions ...... 23 2.3.4 Comparison of CV Results for MEA Electrode and 10 μm CH Electrode in Different K3Fe(CN)6 Solutions ...... 29 vii 2.3.5 Comparisons of SWV Results for MEA Electrode and 10 μm CH Electrode ...... 35

2.4 Conclusions ...... 38

3 STRIPPING ANALYSIS FOR COPPER AND LEAD IONS ...... 39

3.1 Stripping Analysis and Testing Criteria ...... 39 3.2 Experimental ...... 42 3.3 Results and Discussion ...... 44

3.3.1 DPASV Detection of Cu Ion Solutions by MEA Electrode ...... 44 3.3.2 SWASV Detection of Cu Ion Solutions by MEA Electrode ...... 51 3.3.3 DPASV Detection of Pb Ion Solutions by MEA Electrode and 10 µm CH Electrode ...... 53 3.3.4 SWASV Detection of Pb Ion Solution by MEA Electrode and 10 µm CH Electrode ...... 56 3.3.5 Effect of Reversing Potential Direction on Cu and Pb Stripping Analysis ...... 59

3.4 Conclusions ...... 67

4 COUPLING WITH ELECTROCHEMICAL CHIP ANALYZER ...... 69

4.1 Feasibility in Coupling EC‐Chip Analyzer with the MEA Platform ...... 69 4.2 Experimental ...... 70 4.3 Results and Discussion ...... 71

4.3.1 Comparison of K3Fe(CN)6 SWV Results on 2 mm Au Electrode ...... 71 4.3.2 Comparison of SWV Results on Cu Ions Using 2 mm Au Electrode ...... 74 4.3.3 SWV Results on Cu Ions Using the MEA Sensor Platform ...... 78

4.3 Conclusions ...... 79

5 PROJECT CONCLUSION ...... 81

5.1Problem Elucidation ...... 8 1 5.2 Attainment of Project Aims ...... 81

5.2.1 Voltammetric Criterion Design ...... 82 5.2.2 Microelectrode Pattern Design Criterion ...... 82 5.2.3 Array Design Criterion ...... 83 5.2.4 Demonstration and Characterization of the MEA Electrodes ... 83 5.2.5 Application of the MEA Platform to Stripping Analysis ...... 84 5.2.6 Feasibility for Future Coupling of the MEA Platform with EC‐ Chip Analyzer ...... 86

viii 5.3 Further Development Suggestions ...... 87 5.4 Additional Applications of the MEA Sensor Platform ...... 88

6 ENGINEERING APPLICATIONS ...... 90

6.1 Simultaneous Multi‐electrode Detection ...... 90 6.2 Application for On‐site Environmental Monitoring ...... 92

REFERENCES ...... 95

APPENDIX SUPPLEMENTARY INFORMATION ...... 98

A.1 Additional Microfabrication and Sensor Characterization ...... 98 A.2 Additional Stripping Analysis Results for Copper and Lead Ions ...... 102

A.2.1 DPASV on Cu Ion Solutions Utilizing 10 μm Au CH Electrode 1 0 2 A.2.2 SWASV on Cu Ion Solutions Utilizing MEA Electrode ...... 105 A.2.3 SWASV on Cu Ion Solutions Utilizing 10 μm Au CH Electrode ...... 109 A.2.4 DPASV on Pb Ion Solutions Utilizing MEA Electrode ...... 112 A.2.5 DPASV on Pb Ion Solutions Utilizing 10 μm Au CH Electrode 1 1 6 A.2.6 SWASV on Pb Ion Solutions Utilizing MEA Electrode ...... 119 A.2.7 SWASV on Pb Ion Solutions Utilizing 10 μm Au CH Electrode ...... 123 A.2.8 DPASV on Pb Ion Solutions Utilizing MEA Electrode (Reverse Direction) ...... 126 A.2.9 SWASV on Pb Ion Solutions Utilizing MEA Electrode (Reverse Direction) ...... 128 A.2.10 DPASV and SWASV on Mixed Cu & Pb Ion Solutions Utilizing MEA Electrode ...... 130

LIST OF TABLES

Table Page

2.1 ‐ Input parameters for cyclic and square‐wave voltammetry testing...... 20

3.1 ‐ Input parameters for DPASV and SWASV testing of copper, lead, and mixed analytes...... 43

3.2 ‐ Cu peak analysis with DPASV testing and ‐0.1 to 0.3 V scan range...... 49

3.3 ‐ DPASV vs. SWASV for Cu response in Cu and Cu & Pb mixed solutions...... 66

4.1 ‐ Input parameters for SWV testing...... 71

x LIST OF FIGURES

Figure Page

2.1 ‐ Fundamental microelectrode geometries including microdisk (A), square microband (B), and microring (C)...... 10

2.2 ‐ Microelectrode array design platform including entire glass slide array (A), 1 test site with 18 individual microelectrodes (B), and zoomed in view of the electrode pads for 1 test site (C)...... 13

2.3 ‐ Photolithography procedures for fabricating the MEA on glass slide...... 14

2.4 ‐ Half array pictures including: final half array comprising of 9 electrodes, wiring, and electronic contact pads (A), zoomed in views of the 9 electrode test site before and after the final layer of resist is applied and holes are opened up, (B) and (C), respectively...... 1 7

2.5 ‐ Compete array consisting of 18 electrodes, wiring, and electronic contact pads. Three arrays are located on each glass slide...... 17

2.6 ‐ MEA and numbering scheme for completed sensor platform. Numerical designation continued as across wells; Well #2 electrode designations are 10‐18, Well #3 electrode designations are 19‐27...... 18

2.7 ‐ Electrochemical cell configuration during testing of the MEA sensor platform...... 19

2.8 ‐ Comparison of cyclic voltammograms of typical MEA electrode (R10 in this case) and that of a 2

mm macroelectrode on 0.3 mM K3Fe(CN)6 in 0.01 M PBS; MEA electrode: 0.5 to ‐0.1 V, scan rate of 10 mV/s; macroelectrode: 0.6 to ‐0.1 V, scan rate of 10 mV/s. Arrows indicate the potential scanning direction...... 21

2.9 ‐ CV testing of electrode L9 in five successive runs on 0.1 mM K3Fe(CN)6 in 0.01 M PBS, 0.5 to ‐0.1 V with scan rate of 60 mV/s. Arrow indicates the potential scanning direction...... 22

2.10 ‐ Average and 1 standard deviation of L9 electrode tested in 0.01 M PBS, 0.5 to ‐0.1 V with scan rate of 60 mV/s. Arrow indicates the potential scanning direction...... 23

2.11 ‐ CV voltammograms for individual 18 MEA electrodes comprising one test site with 0.3 mM

K3Fe(CN)6 in 0.01 M PBS; 0.5 to ‐0.1 V, scan rate of 10 mV/s...... 25

2.12 ‐ Overlaps of CV curves of all 18 MEA electrodes comprising one test site with 0.3 mM K3Fe(CN)6 in 0.01 M PBS; 0.5 to ‐0.1 V, scan rate of 10 mV/s...... 26

2.13 ‐ Average CV voltammogram and 1 standard deviation for all 18 MEA electrodes comprising one

test site with 0.3 mM K3Fe(CN)6 in 0.01 M PBS; 0.5 to ‐0.1 V, scan rate of 10 mV/s...... 26

2.14 ‐ CV voltammograms for individual 18 MEA electrodes comprising one test site with 0.5 mM

K3Fe(CN)6 in 0.01 M PBS; 0.5 to ‐0.1 V, scan rate of 10 mV/s...... 27 xi 2.15 ‐ Overlaps of CV curves of all 18 MEA electrodes comprising one test site with 0.5 mM K3Fe(CN)6 in 0.01 M PBS; 0.5 to ‐0.1 V, scan rate of 10 mV/s...... 28

2.16 ‐ Average CV voltammogram and 1 standard deviation for 18 MEA electrodes comprising one

test site with 0.5 mM K3Fe(CN)6 in 0.01 M PBS; 0.5 to ‐0.1 V, scan rate of 10 mV/s...... 28

2.17 ‐ Average CV voltammogram and 1 standard deviation comparison for all 18 MEA electrodes

comprising one test site with 0.3 mM and 0.5 mM K3Fe(CN)6 in 0.01 M PBS; 0.5 to ‐0.1 V, scan rate of 10 mV/s...... 29

2.18 ‐ CV curves of an MEA electrode in 0.05, 0.1, 0.3, 0.5, 0.8, and 1.0 mM K3Fe(CN)6 in 0.01 M PBS; 0.5 to ‐0.1 V, scan rate of 10 mV/s. n=1 ...... 32

2.19 ‐ CV calibration curve of MEA electrode in 0.05, 0.1, 0.3, 0.5, 0.8, and 1.0 mM K3Fe(CN)6 in 0.01 M PBS; 0.5 to ‐0.1 V, scan rate of 10 mV/s. n=1 ...... 32

2.20 ‐ Average CV curves of 10 μm CH electrode tested with 0.05, 0.1, 0.3, 0.5, and 1.0 mM K3Fe(CN)6 in 0.01 M PBS; 0.5 to ‐0.1 V, scan rate of 10 mV/s. n=3 ...... 33

2.21 ‐ CV calibration curve of 10 μm CH electrode in 0.05, 0.1, 0.3, 0.5, and 1.0 mM K3Fe(CN)6 in 0.01 M PBS; 0.5 to ‐0.1 V, scan rate of 10 mV/s. n=3 ...... 33

2.22 ‐ Average CV curves of MEA electrode tested with 0.05, 0.1, 0.3, 0.5, and 1.0 mM K3Fe(CN)6 in 0.01 M PBS; 0.5 to ‐0.1 V, scan rate of 10 mV/s. n=3 ...... 34

2.23 ‐ CV calibration curve for MEA electrode in 0.05, 0.1, 0.3, 0.5, and 1.0 mM K3Fe(CN)6 in 0.01 M PBS; 0.5 to ‐0.1 V, scan rate of 10 mV/s. n=3 ...... 34

2.24 ‐ Average SWV responses of MEA electrode L6 tested with 0.05, 0.1, 0.3, 0.5, and 1.0 mM

K3Fe(CN)6 in 0.01 M PBS; 0.5 to ‐0.1 V, scan rate of 10 mV/s. n=3 ...... 35

2.25 ‐ SWV calibration curve for MEA electrode L6 tested in 0.05, 0.1, 0.3, 0.5, and 1.0 mM K3Fe(CN)6 in 0.01 M PBS; 0.5 to ‐0.1 V, scan rate of 10 mV/s. n=3 ...... 36

2.26 ‐ Average SWV responses of 10 μm CH electrode tested with 0.05, 0.1, 0.3, 0.5, and 1.0 mM

K3Fe(CN)6 in 0.01 M PBS; 0.5 to ‐0.1 V, scan rate of 10 mV/s. n=3 ...... 37

2.27 ‐ SWV calibration curve for 10 μm CH electrode tested in 0.05, 0.1, 0.3, and 0.5 mM K3Fe(CN)6 in 0.01 M PBS; 0.5 to ‐0.1 V, scan rate of 10 mV/s. n=3 (response taken as maximum current observed)...... 37

3.1 ‐ Potential‐time sequence for linear (A), staircase (B), differential pulse (C), and square‐wave (D) sweeping stripping modes...... 42

3.2 ‐ MEA electrode L8 DPASV testing (left) and average results with 1 standard deviation (right) on 1 ppb Cu with HNO3; 60 s preconcentration, ‐0.1 to 0.3 V scan range. n=2 ...... 45 xii 3.3 ‐ MEA electrode L8 DPASV testing (left) and average results with 1 standard deviation (right) on

10 ppb Cu with HNO3; 60 s preconcentration, ‐0.1 to 0.3 V scan range. n=3 ...... 45

3.4 ‐ MEA electrode L8 DPASV testing (left) and average results with 1 standard deviation (right) on

20 ppb Cu with HNO3; 60 s preconcentration, ‐0.1 to 0.3 V scan range. n=2 ...... 45

3.5 ‐ MEA electrode L8 DPASV testing (left) and average results with 1 standard deviation (right) on

50 ppb Cu with HNO3; 60 s preconcentration, ‐0.1 to 0.3 V scan range. n=3 ...... 46

3.6 ‐ MEA electrode L8 DPASV testing (left) and average results with 1 standard deviation (right) on

70 ppb Cu with HNO3; 60 s preconcentration, ‐0.1 to 0.3 V scan range. n=3 ...... 46

3.7 ‐ MEA electrode L8 DPASV testing (left) and average results with 1 standard deviation (right) on

100 ppb Cu with HNO3; 60 s preconcentration, ‐0.1 to 0.3 V scan range. n=3 ...... 46

3.8 ‐ MEA electrode L8 DPASV testing (left) and average results with 1 standard deviation (right) on

200 ppb Cu with HNO3; 60 s preconcentration, ‐0.1 to 0.3 V scan range. n=3 ...... 47

3.9 ‐ MEA electrode L8 DPASV testing (left) and average results with 1 standard deviation (right) on

500 ppb Cu with HNO3; 60 s preconcentration, ‐0.1 to 0.3 V scan range. n=3 ...... 47

3.10 ‐ MEA electrode L8 DPASV testing (left) and average results with 1 standard deviation (right) on

1000 ppb Cu with HNO3; 60 s preconcentration, ‐0.1 to 0.3 V scan range. n=3 ...... 47

3.11 ‐ MEA electrode L8 DPASV response on 1 ‐ 1000 ppb Cu ions in HNO3; 60 s preconcentration, ‐ 0.1 to 0.3 V scan range. n=3 ...... 48

3.12 ‐ Average MEA electrode L8 DPASV response, with 1 standard deviation, on 1 ‐ 100 ppb Cu ions

in HNO3; 60 s preconcentration, ‐0.1 to 0.3 V scan range. n=3 ...... 49

3.13 ‐ Cu peak current vs. concentration for MEA electrode L8; DPASV, 60 s preconcentration, ‐0.1 to 0.3 V scan range. n=3 ...... 50

3.14 ‐ Average 10 μm CH electrode DPASV response on 1 ‐ 1000 ppb Cu ions in HNO3; 60 s preconcentration, ‐0.1 to 0.3 V scan range. n=3 ...... 50

3.15 ‐ Average MEA electrode L8 SWASV response on 1 ‐ 1000 ppb Cu ions in HNO3; 60 s preconcentration, ‐0.5 to 0.3 V scan range. n=3 ...... 52

3.16 ‐ Cu peak current vs. concentration for MEA electrode L8; SWASV, 60 s preconcentration, ‐0.5 to 0.3 V scan range. n=3 ...... 52

3.17 ‐ Average 10 μm CH electrode SWASV response on 1 ‐ 1000 ppb Cu ions in HNO3; 60 s preconcentration, ‐0.5 to 0.3 V scan range. n=3 ...... 53

3.18 ‐ Average MEA electrode L8 DPASV response on 1 ‐ 1000 ppb Pb ions in HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 54 xiii 3.19 ‐ Pb peak current vs. concentration for MEA electrode L8; DPASV, 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 55

3.20 ‐ Average 10 μm electrode DPASV response on 1 ‐ 1000 ppb Pb ions in HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 56

3.21 ‐ Average MEA electrode L8 SWASV response on 1 ‐ 1000 ppb Pb ions in HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 57

3.22 ‐ Average 10 μm electrode SWASV response on 1 ‐ 1000 ppb Pb ions in HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 58

3.23 ‐ Average MEA electrode L8 DPASV response on 20 ‐ 500 ppb Pb ions in HNO3; 60 s preconcentration, 0.0 V to ‐0.6 V scan range (reverse potential sweep). n=3...... 60

3.24 ‐ MEA electrode L8 DPASV response vs. concentration on 20 ‐ 500 ppb Pb ions in HNO3; 60 s preconcentration, 0.0 V to ‐0.6 V scan range (reverse potential sweep). n=3...... 60

3.25 ‐ Average MEA electrode L8 SWASV response on 20 ‐ 500 ppb Pb ions in HNO3; 60 s preconcentration, 0.0 V to ‐0.6 V scan range (reverse potential sweep). n=3...... 61

3.26 ‐ MEA electrode L16 DPASV on response on 1 ‐ 200 ppb Cu & Pb ions in HNO3, ‐0.4 V for Pb and 0.3 V for Cu; insert expands Cu responses; 60 s preconcentration, ‐0.6 to 0.4 V scan range. n=363

3.27 ‐ MEA electrode L16 SWASV on response on 1 ‐ 100 ppb Cu & Pb ions in HNO3, ‐0.4 V for Pb and 0.3 V for Cu; insert expands Cu responses; 60 s preconcentration, ‐0.6 to 0.4 V scan range. n=364

3.28 ‐ MEA electrode L16 DPASV on response on Cu peak in 1 ‐ 1000 ppb Cu and Pb mixtures in

HNO3; 60 s preconcentration, ‐0.6 to 0.4 V scan range. n=3 (Expanded insert from Figure 3.26) ...... 64

3.29 ‐ MEA electrode L16 DPASV current response vs. concentration at the Cu peak for 1 ‐ 1000 ppb 2 Cu and Pb mixtures in HNO3 at the Cu peak; 60 s preconcentration, ‐0.6 to 0.4 V scan range. R = 0.920 (1‐70 Pb)...... 65

3.30 ‐ MEA electrode L16 SWASV on response on Cu peak in 1 ‐ 1000 ppb Cu and Pb mixtures in

HNO3 ; 60 s preconcentration, ‐0.6 to 0.4 V scan range...... 65

3.31 ‐ MEA electrode L16 SWASV current response vs. concentration at the Cu peak for 1 ‐ 1000 ppb 2 Pb ions in HNO3 at the Cu peak; 60 s preconcentration, ‐0.6 to 0.4 V scan range. R = 0.940 (1‐70 Pb)...... 66

4.1 ‐ Average SWV responses of 2 mm gold working electrode tested in triplicate with 0.05, 0.1, 0.3,

0.5, and 1.0 mM K3Fe(CN)6 in 0.01 M PBS; 0.5 to ‐0.1 V, recorded with the EC‐Chip analyzer. .... 72

4.2 ‐ Average SWV responses of 2 mm gold working electrode tested in triplicate with 0.05, 0.1, 0.3,

0.5, and 1.0 mM K3Fe(CN)6 in 0.01 M PBS; 0.5 to ‐0.1 V, recorded with the CHI 1220 analyzer. . 72 xiv 4.3 ‐ Peak current vs. concentration for 2mm gold working electrode SWV testing of K3Fe(CN)6 in 0.01 M PBS; 0.5 to ‐0.1 V, recorded with the EC‐Chip analyzer...... 73

4.4 ‐ Peak current vs. concentration for 2mm gold working electrode SWV testing of K3Fe(CN)6 in 0.01 M PBS; 0.5 to ‐0.1 V, recorded with the CHI 1220 analyzer...... 74

4.5 ‐ Average SWV responses of 2 mm gold working electrode tested in triplicate with 100, 300, 500,

700, and 1000 ppb Cu in HNO3; 0.5 to ‐0.1 V, recorded with the EC‐Chip analyzer...... 75

4.6 ‐ Average SWV responses of 2 mm gold working electrode tested in triplicate with 100, 300, 500,

700, and 1000 ppb Cu in HNO3; 0.5 to ‐0.1 V, recorded with the CHI 1220 analyzer...... 76

4.7 ‐ Comparison of Average SWV responses of 2 mm gold working electrode tested in triplicate with

300 and 1000 ppb Cu in HNO3; 0.5 to ‐0.1 V, recorded with the CHI 1220 and EC‐Chip analyzers...... 77

4.8 ‐ Comparison of Average SWV responses of 2 mm gold working electrode tested in triplicate with

20 ppm Cu in HNO3; 0.5 to ‐0.1 V, recorded with the CHI 1220 and EC‐Chip analyzers...... 77

4.9 ‐ Average SWV responses of MEA electrode L8 tested in triplicate with 100, 300, 500, 700, and

1000 ppb Cu in HNO3; 0.5 to ‐0.1 V, recorded with the CHI 1220 analyzer...... 78

6.1 – Schematic illustration of simultaneous individual 3 electrode stripping analysis for Cu and Pb ion utilizing the MEA sensor platform...... 92

A.1 ‐ Complete fabricated MEA sensor (left) and partially completed sensor (right)...... 98

A.2 ‐ Nine‐electrode array constituting half of one test site on the MEA sensor platform; before resist layer (left) and after resist layer is applied (right)...... 98

A.3 ‐ Nine‐electrode array constituting half of one test site on the MEA sensor platform after resist layer is applied (left) and with approximate dimensions (right)...... 99

A.4 ‐ Nine‐electrode array (before resist mask layer) with approximate dimensions of base sensor pads and wireing...... 99

A.5 ‐ Electrode after resist mask layer is applied (left) and with approximate dimensions (right). ... 100

A.6 ‐ Wiring junction above electronic contact pads (left) and with wire dimensions (right)...... 100

A.7 ‐ Nine‐electrode array composing half of one test site on the MEA sensor platform...... 100

A.8 ‐ One test site on the MEA sensing platform consisting of 18 individual microelectrodes in the array...... 101

A.9 ‐ Complete MEA sensing platform with 96 well plate future prototypes will be designed for...... 101

A.10 ‐ 10 μm CH electrode L8 DPASV testing (left) and average results with 1 standard deviation

(right) on 1 ppb Cu with HNO3; 60 s preconcentration, ‐0.1 to 0.3 V scan range. n=3 ...... 102 xv A.11 ‐ 10 μm CH electrode L8 DPASV testing (left) and average results with 1 standard deviation

(right) on 10 ppb Cu with HNO3; 60 s preconcentration, ‐0.1 to 0.3 V scan range. n=3 ...... 102

A.12 ‐ 10 μm CH electrode L8 DPASV testing (left) and average results with 1 standard deviation

(right) on 20 ppb Cu with HNO3; 60 s preconcentration, ‐0.1 to 0.3 V scan range. n=3 ...... 103

A.13 ‐ 10 μm CH electrode L8 DPASV testing (left) and average results with 1 standard deviation

(right) on 50 ppb Cu with HNO3; 60 s preconcentration, ‐0.1 to 0.3 V scan range. n=3 ...... 103

A.14 ‐ 10 μm CH electrode L8 DPASV testing (left) and average results with 1 standard deviation

(right) on 70 ppb Cu with HNO3; 60 s preconcentration, ‐0.1 to 0.3 V scan range. n=3 ...... 103

A.15 ‐ 10 μm CH electrode L8 DPASV testing (left) and average results with 1 standard deviation

(right) on 100 ppb Cu with HNO3; 60 s preconcentration, ‐0.1 to 0.3 V scan range. n=3 ...... 104

A.16 ‐ 10 μm CH electrode L8 DPASV testing (left) and average results with 1 standard deviation

(right) on 200 ppb Cu with HNO3; 60 s preconcentration, ‐0.1 to 0.3 V scan range. n=3 ...... 104

A.17 ‐ 10 μm CH electrode L8 DPASV testing (left) and average results with 1 standard deviation

(right) on 500 ppb Cu with HNO3; 60 s preconcentration, ‐0.1 to 0.3 V scan range. n=3 ...... 104

A.18 ‐ 10 μm CH electrode L8 DPASV testing (left) and average results with 1 standard deviation

(right) on 1000 ppb Cu with HNO3; 60 s preconcentration, ‐0.1 to 0.3 V scan range. n=3 ...... 105

A.19 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 1 ppb Cu with HNO3; 60 s preconcentration, ‐0.5 to 0.3 V scan range. n=3 ...... 105

A.20 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 10 ppb Cu with HNO3; 60 s preconcentration, ‐0.5 to 0.3 V scan range. n=3 ...... 106

A.21 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 20 ppb Cu with HNO3; 60 s preconcentration, ‐0.5 to 0.3 V scan range. n=3 ...... 106

A.22 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 50 ppb Cu with HNO3; 60 s preconcentration, ‐0.5 to 0.3 V scan range. n=3 ...... 106

A.23 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 70 ppb Cu with HNO3; 60 s preconcentration, ‐0.5 to 0.3 V scan range. n=3 ...... 107

A.24 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 100 ppb Cu with HNO3; 60 s preconcentration, ‐0.5 to 0.3 V scan range. n=2 ...... 107

A.25 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 200 ppb Cu with HNO3; 60 s preconcentration, ‐0.5 to 0.3 V scan range. n=3 ...... 107

A.26 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 500 ppb Cu with HNO3; 60 s preconcentration, ‐0.5 to 0.3 V scan range. n=3 ...... 108 xvi A.27 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 1000 ppb Cu with HNO3; 60 s preconcentration, ‐0.5 to 0.3 V scan range. n=3 ...... 108

A.28 ‐ 10 μm CH electrode L8 SWASV testing (left) and average results with 1 standard deviation

(right) on 1 ppb Cu with HNO3; 60 s preconcentration, ‐0.5 to 0.3 V scan range. n=3 ...... 109

A.29 ‐ 10 μm CH electrode L8 SWASV testing (left) and average results with 1 standard deviation

(right) on 10 ppb Cu with HNO3; 60 s preconcentration, ‐0.5 to 0.3 V scan range. n=3 ...... 109

A.30 ‐ 10 μm CH electrode L8 SWASV testing (left) and average results with 1 standard deviation

(right) on 20 ppb Cu with HNO3; 60 s preconcentration, ‐0.5 to 0.3 V scan range. n=3 ...... 110

A.31 ‐ 10 μm CH electrode L8 SWASV testing (left) and average results with 1 standard deviation

(right) on 50 ppb Cu with HNO3; 60 s preconcentration, ‐0.5 to 0.3 V scan range. n=3 ...... 110

A.32 ‐ 10 μm CH electrode L8 SWASV testing (left) and average results with 1 standard deviation

(right) on 70 ppb Cu with HNO3; 60 s preconcentration, ‐0.5 to 0.3 V scan range. n=3 ...... 110

A.33 ‐ 10 μm CH electrode L8 SWASV testing (left) and average results with 1 standard deviation

(right) on 100 ppb Cu with HNO3; 60 s preconcentration, ‐0.5 to 0.3 V scan range. n=3 ...... 111

A.34 ‐ 10 μm CH electrode L8 SWASV testing (left) and average results with 1 standard deviation

(right) on 200 ppb Cu with HNO3; 60 s preconcentration, ‐0.5 to 0.3 V scan range. n=3 ...... 111

A.35 ‐ 10 μm CH electrode L8 SWASV testing (left) and average results with 1 standard deviation

(right) on 500 ppb Cu with HNO3; 60 s preconcentration, ‐0.5 to 0.3 V scan range. n=3 ...... 111

A.36 ‐ 10 μm CH electrode L8 SWASV testing (left) and average results with 1 standard deviation

(right) on 100 ppb Cu with HNO3; 60 s preconcentration, ‐0.5 to 0.3 V scan range. n=3 ...... 112

A.37 ‐ MEA electrode L8 DPASV testing (left) and average results with 1 standard deviation (right) on

1 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=6 ...... 112

A.38 ‐ MEA electrode L8 DPASV testing (left) and average results with 1 standard deviation (right) on

10 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 113

A.39 ‐ MEA electrode L8 DPASV testing (left) and average results with 1 standard deviation (right) on

20 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 113

A.40 ‐ MEA electrode L8 DPASV testing (left) and average results with 1 standard deviation (right) on

50 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 113

A.41 ‐ MEA electrode L8 DPASV testing (left) and average results with 1 standard deviation (right) on

70 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=2 ...... 114

A.42 ‐ MEA electrode L8 DPASV testing (left) and average results with 1 standard deviation (right) on

100 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 114 xvii A.43 ‐ MEA electrode L8 DPASV testing (left) and average results with 1 standard deviation (right) on

200 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 (unsuccessful) .... 114

A.44 ‐ MEA electrode L8 DPASV testing (left) and average results with 1 standard deviation (right) on

500 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=2 ...... 115

A.45 ‐ MEA electrode L8 DPASV testing (left) and average results with 1 standard deviation (right) on

1000 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=2 ...... 115

A.46 ‐ 10 μm CH electrode L8 DPASV testing (left) and average results with 1 standard deviation

(right) on 1 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 116

A.47 ‐ 10 μm CH electrode L8 DPASV testing (left) and average results with 1 standard deviation

(right) on 10 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 116

A.48 ‐ 10 μm CH electrode L8 DPASV testing (left) and average results with 1 standard deviation

(right) on 20 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 117

A.49 ‐ 10 μm CH electrode L8 DPASV testing (left) and average results with 1 standard deviation

(right) on 50 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 117

A.50 ‐ 10 μm CH electrode L8 DPASV testing (left) and average results with 1 standard deviation

(right) on 70 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 117

A.51 ‐ 10 μm CH electrode L8 DPASV testing (left) and average results with 1 standard deviation

(right) on 100 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 118

A.52 ‐ 10 μm CH electrode L8 DPASV testing (left) and average results with 1 standard deviation

(right) on 200 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 118

A.53 ‐ 10 μm CH electrode L8 DPASV testing (left) and average results with 1 standard deviation

(right) on 500 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 118

A.54 ‐ 10 μm CH electrode L8 DPASV testing (left) and average results with 1 standard deviation

(right) on 1000 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 119

A.55 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 1 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 119

A.56 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 10 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 120

A.57 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 20 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 120

A.58 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 50 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 120 xviii A.59 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 70 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 121

A.60 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 100 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 121

A.61 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 200 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 (unsuccessful) ...... 121

A.62 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 500 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 122

A.63 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 1000 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=2 ...... 122

A.64 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 1 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 123

A.65 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 10 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 123

A.66 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 20 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 124

A.67 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 50 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 124

A.68 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 70 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 124

A.69 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 100 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 125

A.70 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 200 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=2 ...... 125

A.71 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 500 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 125

A.72 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 1000 ppb Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.0 V scan range. n=3 ...... 126

A.73 ‐ MEA electrode L8 DPASV testing (left) and average results with 1 standard deviation (right) on

20 ppb Pb with HNO3; 60 s preconcentration, 0.0 to ‐0.6 V scan range. n=3 (reverse direction) ...... 126 xix A.74 ‐ MEA electrode L8 DPASV testing (left) and average results with 1 standard deviation (right) on

50 ppb Pb with HNO3; 60 s preconcentration, 0.0 to ‐0.6 V scan range. n=3 (reverse direction) ...... 127

A.75 ‐ MEA electrode L8 DPASV testing (left) and average results with 1 standard deviation (right) on

100 ppb Pb with HNO3; 60 s preconcentration, 0.0 to ‐0.6 V scan range. n=3 (reverse direction) ...... 127

A.76 ‐ MEA electrode L8 DPASV testing (left) and average results with 1 standard deviation (right) on

500 ppb Pb with HNO3; 60 s preconcentration, 0.0 to ‐0.6 V scan range. n=3 (reverse direction) ...... 127

A.77 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 20 ppb Pb with HNO3; 60 s preconcentration, 0.0 to ‐0.6 V scan range. n=3 (reverse direction) ...... 128

A.78 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 50 ppb Pb with HNO3; 60 s preconcentration, 0.0 to ‐0.6 V scan range. n=3 (reverse direction) ...... 128

A.79 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 100 ppb Pb with HNO3; 60 s preconcentration, 0.0 to ‐0.6 V scan range. n=3 (reverse direction) ...... 129

A.80 ‐ MEA electrode L8 SWASV testing (left) and average results with 1 standard deviation (right)

on 500 ppb Pb with HNO3; 60 s preconcentration, 0.0 to ‐0.6 V scan range. n=3 (reverse direction) ...... 129

A.81 ‐ MEA electrode L8 DPASV testing (left) and SWASV testing (right) on 1 ppb Cu & Pb with HNO3; 60 s preconcentration, ‐0.6 to 0.4 V scan range. n=3 ...... 130

A.82 ‐ MEA electrode L8 DPASV testing (left) and SWASV testing (right) on 10 ppb Cu & Pb with

HNO3; 60 s preconcentration, ‐0.6 to 0.4 V scan range. n=3 ...... 130

A.83 ‐ MEA electrode L8 DPASV testing (left) and SWASV testing (right) on 20 ppb Cu & Pb with

HNO3; 60 s preconcentration, ‐0.6 to 0.4 V scan range. n=3 ...... 131

A.84 ‐ MEA electrode L8 DPASV testing (left) and SWASV testing (right) on 50 ppb Cu & Pb with

HNO3; 60 s preconcentration, ‐0.6 to 0.4 V scan range. n=3 ...... 131

A.85 ‐ MEA electrode L8 DPASV testing (left) and SWASV testing (right) on 70 ppb Cu & Pb with

HNO3; 60 s preconcentration, ‐0.6 to 0.4 V scan range. n=3 ...... 131

A.86 ‐ MEA electrode L8 DPASV testing (left) and SWASV testing (right) on 100 ppb Cu & Pb with

HNO3; 60 s preconcentration, ‐0.6 to 0.4 V scan range. n=3 ...... 132

A.87 ‐ MEA electrode L8 DPASV testing (left) and SWASV testing (right) on 200 ppb Cu & Pb with

HNO3; 60 s preconcentration, ‐0.6 to 0.4 V scan range. n=3 ...... 132 xx A.88 ‐ MEA electrode L8 DPASV testing (left) and SWASV testing (right) on 500 ppb Cu & Pb with

HNO3; 60 s preconcentration, ‐0.6 to 0.4 V scan range. n=3 ...... 132

A.89 ‐ MEA electrode L8 DPASV testing (left) and SWASV testing (right) on 1000 ppb Cu & Pb with

HNO3; 60 s preconcentration, ‐0.6 to 0.4 V scan range. n=3 ...... 133

1 CHAPTER I

PROJECT INTRODUCTION

1.1 Problem Description

Charged species identification and quantification is very important to analytical chemistry experiments in industrial, environmental, and academic experimentation. Many systems and environments require testing for trace levels of ions or molecules that reside, contaminate, or transform through some reaction. Salinity is an important criterion of water control both in agricultural and municipal water supplies. Salts are strong electrolytes consisting of metal cations and counter ions that disassociate in aqueous solutions. These metal cation contaminants such as copper, lead, zinc, and cadmium can be present in foods, beverages, drinking water, and aquatic environments.1‐4 High levels of

charged species raise levels of concern and, therefore, accurately monitoring or quantifying them is desired.

Traditionally environment or industrial process samples are taken on site and

removed to laboratory facilities where electrochemical, chromatographic, and spectroscopic

methods are employed to detect, observe, and quantify any ions or ionic species within the

sample. Examples of typical methods utilized include inductively coupled plasma ‐ mass

spectrometry (ICP‐MS), differential pulse anodic stripping Voltammetry (DPASV), graphite

furnace atomic absorption spectrometry (GF‐AAS), cold vapor atomic absorption

spectrometry (CV‐AAS), and cold vapor atomic fluorescence spectrometry (CV‐AFS).5‐7

However, these test methods vary in sensitivity to the charged species of interest

(dependent on method employed), take an undesirable amount of time to obtain results, and the samples have a greater probability of contamination or loss due to the handling and transport prior to testing.1,8 Advantages of new analytical tools that are capable of real 2 time process monitoring or real time environmental monitoring include improved process control, minimization of environmental impact, and ability for continuous monitoring with options for early detection.1,2 Where, the greatest advantage is the rapid return on the test results, enabling quick action responses.

Charged species of concern can be monitored on site with an appropriately designed sensor capable of all of the aforementioned advantages. Additionally, due to advances and trends towards cleaner (green) analytical chemistry, a new sensor system should be capable of monitoring an environment without polluting the system or affecting it in any adverse manner.1 This can be a main constraint parameter on new sensor system development and must be considered.

This thesis is the summary of the work completed to develop a low cost reliable sensor that is sensitive and selective to the heavy metal ions, including copper and lead, which act as contaminants in aqueous systems. Parameters and characterization of the sensor are proven along with the operating theory and justification of design and usage.

1.2 Overview of the Solution

Electrochemical (EC) methods offer great promise for ion detection. Features like

independence from an optical path length and high sensitivity, approaching that of

fluorescence, along with low power requirements, low cost, ability to be miniaturized, and

adaptability with advanced micromachining and microfabrication technologies, make EC

method very appealing.1 Voltammetry, one of the most widely used electrochemical methods in the detection of ions, is used to examine the kinetics of electrode processes for which current is monitored as the applied potential is changed. Alternatively chronopotentiometry, another electrochemical method, explores the kinetics of electrode processes for which the potential is monitored as the current is changed; this method is not 3 explored herein. The overpotential measured can be directly related to the concentration of the analyte in solution. Traditionally, sensor electrodes used in voltammetric and/or chronopotentiometric experiments have typically been relatively large electrodes on the millimeter scale. However, since the advent of microelectrodes, there is no longer a reason to rely on bulky electrodes or difficult electrochemical cells; fast, easy, environmentally friendly electrochemical systems can be obtained.1

1.2.1 Stripping Voltammetry Analysis

Stripping analysis has proven extremely powerful for identification of trace metals

in environmental, clinical, or industrial samples.1,4,9,10 Due to the coupling of a

preconcentrating step with a sensitive electrochemical measurement technique, higher

sensitivity can be obtained.1,4,9 Furthermore, since stripping analysis utilizes only low power and the instrumentation needed for implementation is small and portable, its feasibility increases as a reliable on‐site trace metal sensing system.1 As an example,

Giacomino reports, in a 2008 study on the parameters affecting determination of mercury using anodic stripping voltammetry on a gold electrode, that the US Environmental

Protection Agency (EPA) has recommended adopting stripping analysis for the

quantification of mercury.6

Anodic stripping voltammetry (ASV) is a form of stripping voltammetry where an

under potential is set for a period of time, the preconcentrating step, allowing the positive

charged metal ions to be reduced and electroplated on the electrode surface. Then the

potential is swept towards an overpotential effectively oxidizing the metal species, releasing

the ions and electrons from the electrode surface. This preconcentration effectively

increases the amount of metal ions at the electrode surface, with respect to the

concentration in solution, and allows a favorable signal‐to‐background ratio.4 Current is 4 monitored during a second phase sweeping step and is dependent on the concentration of charged species accumulated at the electrode and the time preconcentrating is allowed.3

The ASV technique utilizes a three electrode test method where a working electrode is used to monitor the oxidation‐reduction reaction, a counter electrode is used to complete the circuit, and a reference electrode is used for comparison. Typically reference electrodes and counter electrodes are silver/silver chloride and platinum wire, respectively. However there are a number of different types of both electrodes.

The working electrode (test electrode) has been proven to strongly influence the performance of the electrochemical measurements. Historically, mercury electrodes worked very well due to a renewable, reproducible, and smooth surface.1,4 However

because of the toxicity concerns with mercury, a suitable alternative needs to be

incorporated in the working electrode design. Alternative metals have been utilized, such

as gold or platinum, and results have been presented for both elements.3,6,8,9,11‐14

Additionally many experiments have favorable outcomes utilizing gold working electrodes over other element choices.3,6,8,9,12,13

1.2.2 Square Wave Anodic Stripping Voltammetry (SWASV) and Differential Pulse Anodic Stripping Voltammetry (DPASV)

As previously mentioned, the second step in stripping analysis involves a sweeping

of the potential from an under potential to an over potential state. Sweeping techniques

including linear, staircase, square wave, and pulse methods have been used.3,5,6,8,12‐15 Higher

sensitivities have been reported depending on the sweeping method and the environment

the test is being performed with.6 Square wave voltammetry (SWV) and differential pulse

voltammetry (DPV) are derivatives of linear sweep and staircase where current is monitored while the potential between a reference electrode (example Ag/AgCl) and a 5 working electrode is swept in a series of stair steps with a square wave or a series of regular voltages superimposed on stair steps. These advanced sweeping methods offer greater sensitivity than linear sweep or staircase sweep.6,8,13 When combined with the preconcentrating step in anodic stripping analysis these methods are named square wave anodic stripping voltammetry (SWASV) and differential pulse anodic stripping voltammetry

(DPASV). Furthermore, these methods have been proven with heavy metal ion detection in aqueous systems.1‐4,6,8,10,12,13,15‐21

1.2.3 Microelectrode and Nonlinear Diffusion

Electrochemical testing can further be improved by utilizing microelectrodes in

place of traditional electrodes. Microelectrodes are defined to be electrodes that have

micrometer (μm) dimension; in contrast, traditional electrodes are on the millimeter (mm)

scale or larger. Miniaturization of electrodes offers many practical and fundamental

advantages, namely, reduction of resistance (ohmic drop), reduction of sample

consumption, ability to incorporate many electrodes in a small area, and greater ability to

facilitate measurements in low‐ionic‐strength water samples.1 The foremost advantage to

using this small sized electrode is in the mass transport uniqueness from the nonlinear

diffusion properties accompanying it and this advantage is further amplified when multiple

electrodes are utilized in an array. Nonlinear diffusion happens at the boundary of the

electrode and from the increased perimeter‐to‐surface area ratio exhibited by

microelectrodes, with respect to larger traditional electrodes, current amplification is

accomplished.3,11,14,22‐26 Extensive work has been done to show that microdisk design

(circular disk shaped) have hemispherical diffusion patterns and microband design have hemispherical or a combination hemicylindrical‐hemispherical diffusion pattern whether they are square or rectangular shaped, respectively.3,22,25,26 Diffusion layers are formed next 6 to the electrode surface and electroactive analytes must pass through this layer to reach the

electrode. These diffusion patterns are established within seconds and are significant due

to the radial component of the diffusion; greater amounts of electroactive analyte particles

per surface area travel to the electrode within a set amount of time.26 Furthermore 3‐D

(hemispherical) diffusion associated with microdisk or micro‐square electrodes have a

steady state mass flux to the electrode surface resulting in a sigmoidal cyclic

voltammograms instead of peak shaped voltammograms that would be observed with

traditional or non‐square microband electrodes.3,26 With their small size, larger perimeter‐ to‐surface area ratio, and nonlinear radial diffusion through the diffusion zones greater current density is possible. This leads to larger response changes during testing and improves signal‐to‐noise ratios, which is by definition is an increase in sensitivity.

By utilizing microelectrodes in an array fashion, one can capitalize on the enhanced properties the smaller microelectrode size contributes and also further diversity and flexibility by having a platform that can be utilized in multiple microelectrode combinations.

One can simply use all microelectrodes, set at different potential ranges (assuming voltammetry electrochemical method is performed), use the microelectrodes as a census electrode with all electrodes performing as one capitalizing on their individual enhancements but allowing greater current flow which could be beneficial if the current monitoring instrumentation is not capable of small currents (1 – 1000 pA), or some situational unique combination of the two former scenarios. However, there is a fundamental criterion associated with microelectrodes in an array design. Namely, inter‐ electrode spacing must be such that diffusion zones from the individual microelectrodes don’t overlap and constructively interfere to produce planar diffusion zones much like those associated with larger electrodes. Extensive studies comparing inter‐electrode spacing with 7 current responses reportedly use or suggest using an inter‐electrode spacing of ten times the width or diameter of the electrode.2,3,26‐28

1.2.4 Combination of Voltammetry and Microelectrode

By utilizing an electrochemical testing method coupled to microelectrodes in an

array design, trace levels of heavy metal ions can be detected. Advantages with

preconcentrating, associated with stripping analysis, and increased current density

characteristics from the microelectrodes should allow sensing in the low part‐per‐billion

(ppb) range. Furthermore, due to the small size of microelectrode arrays and the small

instrumentation requirements a portable, environmentally friendly, and simple sensing

system can be designed.

1.3 Aims

This thesis is the summary of the work completed to develop a low cost reliable

sensor that is sensitive and selective to the heavy metal ions, copper and lead, that act as

contaminants in aqueous systems. The goals of this project are:

(i) Design and fabricate a new sensor platform to perform voltammetric

measurements.

(ii) Design a microelectrode pattern (microdisk, microband, or micro‐ring) that

maximizes current density at the electrode surface and improves signal‐to‐noise

ratio and sensitivity.

(iii) Incorporate multiple microelectrodes in an array design to allow detection of

multiple measurable analytes in one aqueous solution and to allow comparison

of same solution responses between multiple electrodes. 8 (iv) Prove function and characterize the microelectrode sensing system in K3Fe(CN)6

electrochemical standard.

(v) Demonstrate the feasibility of using the microelectrode sensing system to detect

copper and lead ions in aqueous solutions.

(vi) Evaluate the potential of future coupling the microelectrode sensing system

with a homemade square wave voltammetry device.

1.4 Outline of Technical Contents

The remainder of this thesis focuses on the fabrication, characterization, application, and future potential of a prototype platform microelectrode array sensing system for water quality monitoring. The information is organized into design, fabrication, proof, and characterization (chapter 2), application with ASV for copper and lead ion detection in aqueous solutions (chapter 3), and feasibility of future coupling with a homemade electrochemical‐chip analyzer (chapter 4). Each technical chapter will include introductory and concluding sections pertinent to the information conveyed within the chapter.

Following the technical chapters is a concluding chapter, discussing future applications and related work, and an engineering applications chapter further discussing simultaneous individual electrode experimentation, along with, advanced discussion of on‐site

environmental monitoring. Ending the thesis is an Appendix containing additional

information on the fabrication and application of the sensor platforms. 9 CHAPTER 2

DESIGN AND CHARACTERIZATION

2.1 Voltammetric Criteria for Testing Utilizing Microelectrodes

When using Anodic Stripping Voltammetry (ASV) as the electrochemical testing

method for heavy metal ion detection, sensitivity is greatly increased due to the

preconcentrating step the method employs.4 Furthermore, sensitivity and testing versatility is further enhanced when the preconcentrating step is coupled with microelectrodes in an array fashion. However, great care needs to be taken in the design of the microelectrodes and microelectrode arrays to constructively enhance sensitivity and versatility.

Microelectrode array design is most complicated by criteria involved with the microelectrode parameters. The benefits and current amplification from microelectrodes needed to be realized and diversity with an array is desirable, but size, shape, composition, fabrication technique, and interelectrode spacing needs to be specified. These parameters were investigated and design rational for each is given in the following discussion.

Compact size, high sensitivity, low cost, and high repeatability are of great importance in sensor fabrication, especially when multiple electrodes comprise an array.

The diversity of testing that can be performed is expanded with all of the electrodes having same shape, size, composition and characteristics. One can only expect comparable responses between electrodes to be correlated if the electrodes are identical. This makes the photolithography technique, utilized in silicon semiconductor industry, very appealing for microelectrode array fabrication. The process of photolithography involves constructing masks and utilizing ultraviolet light to photoactively change a resist polymer.

Thus through development and etching (layer removal), microfabricated devices are made. 10 This fabrication technique is very repeatable and one can develop any electrode pattern desired by simply modifying the photomasks that pattern the microdevices. Final products, being on the low micron scale (limited to 1‐5 μm), have reproducible and well defined

geometries exhibiting near identical chemical and physical performance

characteristics.2,3,10,14,26,27 Other methods of microelectrode construction have been

reported, an example being conductive wire in a capillary tube that is heated to seal the

glass around the wire, or epoxy in glass holding a conducting wire, and screen printed

microdevices but these methods are not as repeatable or arrays are more difficult to

construct than by lithography processes.14

Size and shape of a microelectrode are also very important. Thormann stated in

1985 that microelectrodes fabricated with lithography techniques similar to the design we

are attempting in this thesis are size limited to less than or equal to 10 μm width or

diameter.14 Technological advancement has pushed the size limitations to much less than

10 μm for lithography fabrication. However, for literature comparison reasons we choose to match the 10 μm sized electrodes for the initial protocol sensor platform. Most microelectrodes have one of three different fundamental shapes: microdisk, microband, or microring shapes, shown in Figure 2.1.

A B C

Figure 2.1 Fundamental microelectrode geometries including microdisk (A), square microband (B), and microring (C).

Literature review has indicated that nonlinear, 3‐D, hemispherical diffusion is

prevalent in microdisk, microring, and square microband electrode shapes.2,3,14,22,24‐26 11 Hemispherical 3‐D diffusion has steady state sigmoidal cyclic voltammograms from the steady state mass flux of electroactive analytes to the sensor surface.26 This nonlinear

diffusion mass transport enhancement, over traditional electrodes, leads to response

amplification and greater sensitivity. Additionally, further literature evidence explains that

current flow is a function of perimeter‐to‐area ratio (P/A) and indicates that the microring

has the best response over long testing time.24 However, over short times, microband

geometry will outperform microring geometries with square microband geometries

maximizing performance.3,22,24,25 On an aside, when utilizing photolithography to micromachine a sensor platform, masks must be generated to photoactively react a resist polymer. Square mask geometries are much easier to make, compared to circular geometries, and greater precision is accomplished by using them.

The important parameter with utilizing microelectrodes in an array format is the interelectrode spacing. If the electrodes are close packed, the diffusion zones formed in the first few seconds of testing can interfere resulting in overlap which destroys the nonlinear diffusion enhancement from the microelectrodes.2,3,26 An array with closely packed

electrodes will perform like one large traditional electrode (macroelectrode), while an array

of loosely packed electrodes, will perform closely to an ideal state of multiple responses of a

single microelectrode.2 It has been suggested that using an interelectrode spacing of 10x the width or diameter of the microelectrode is considered to be loosely packed.2,3,27,28

Traditionally ASV is performed with a mercury based electrode, thin mercury film electrode and mercury hanging drop as examples, but due to the environmental and toxic concerns with mercury an alternative solid element electrode composition is desirable.

Studies have shown platinum or gold to be candidates for electrodes and many argue that gold has better performance over platinum.1,3,6,8‐14 Alternative compositions have been suggested including bismuth‐film, carbon, and silver.1,10 Due to literature review suggesting 12 gold to be superior to platinum and silver for solid single element electrodes, it was deemed the best candidate for ASV testing of heavy metal ions.8

Final parameters that need to be addressed are the composition of the mask layer that shields the electrode wiring and electrode position with respect to this mask layer.

There are a variety of mask layers that can be utilized in this type of sensor platform including oxide coatings (silicon oxide), silicon nitride, or a simple resist coating.14,23,28 Due

to the fabrication simplicity and 1981 work by AOKI, resist polymer coating was deemed

advantageous over other coatings while silicon nitride would be less susceptible to

corrosion.23,28 Furthermore, the most simplistic fabrication approach would be to open holes in the resist mask layer directly above the electrodes and then to utilize plasma etch to clean the electrode surface. This approach was performed on a traditional electrode

(macroelectrode) but a literature search failed to show where it was done on a microelectrode array.14,23 Work has been done to model the transient current response through recessed microelectrodes. While there is not as much current that flows through recessed compared to raised or flat microelectrodes there still is nonlinear diffusion which amplifies the current density increasing sensitivity.22

The result from the aforementioned advantages in stripping analysis and microelectrode array performance lead to the development of a simplistic prototype sensor platform utilizing square gold microelectrodes in an array with 10x the width for interelectrode spacing. This sensor platform is tested with cyclic voltammetry to demonstrate 3‐D hemispherical steady state sigmoidal voltammograms, compared with a commercially purchased 10 μm microdisk electrode, and utilized with ASV in heavy metal

ion analysis for aqueous environments. Figure 2.2 is the design schematic for

microelectrode array platform that is used in this thesis work. Part A displays the

electrodes, wiring, and electronic contact pads placement on the glass slide. Part B is an 13 expanded view of 1 test site showing electrodes, wiring, and electronic contact pads. Part C is an expanded view of 1 test site showing the electrodes and wiring connections. The large red bands seen in the top and bottom of part A are for glass slide alignment. Note Figure 2.2 shows the initial gold layer before a final layer of resist is applied and holes are opened up above the electrode contact pads, defining the true electrode, and the electronic contact pads.

A B

Figure 2.2 Microelectrode array design platform including entire glass slide array (A), 1 test site with 18 individual microelectrodes (B), and zoomed in view of the electrode pads for 1 test site (C).

14 2.2 Experimental

2.2.1 Photolithography Fabrication of MEA

Microelectrode arrays (MEA) sensors were constructed by implementing basic photolithography methodology utilized in integrated circuit fabrication. Glass slides were

acquired and through electron beam deposition a thin layer of titanium, approximately 150

nm, was adhered to the exterior glass surface to act as a bonding agent intermediate between

the glass elements and the much thicker gold layer, approximately 1 μm thick, part A‐C in Figure

2.3. Layer thickness was monitored with a piezoelectric quartz crystal microbalance based thickness monitor. Entire gold‐coated slides were stripped to exact geometries specified for 54

individually isolated electrodes with the photolithography technique; number of electrodes was

convenient for prototype platform. Figure 2.3 shows the schematic process for the general

photolithography technique utilized in sensor fabrication that is summarized as follows:

Figure 2.3 Photolithography procedures for fabricating the MEA on glass slide.

1. Fresh gold‐coated glass slides were used as substrates, and Photoresist 1813 was

applied to thickness by spinning 40 seconds at 2000 rpm on a spin coater, part D in 15 Figure 2.3. This was done directly after gold surface sputtering for clean surface

assurance. The photoresist was heat set on the slide surface by heating to 95°C for 90

seconds on a general hot plate.

2. The slide is then exposed on a mask aligner to light from a mercury lamp at 365 nm

wavelength for 8 seconds. Submerging in developer 352 for 1 minute develops and

removes the exposed part of the photoresist. The slide is then cleaned with deionized

water and dried with a nitrogen gun to stop development reaction on the photoresist.

3. The gold layer is removed from everywhere exposed to UV light with iodine based gold

etch followed with a HF bath using buffered oxide etch (BOE) to remove titanium layer

beneath, part E in Figure 2.3. These etch steps were completed quickly to avoid

overexposure leading to sensor damage. The slide is then cleaned with consecutive

rinses in acetone, ethanol, methanol, deionized water, and then dried with a nitrogen

gun. This left all contact pads, circuitry, and electrode pads on the slides.

4. Further cleaning was completed with oxygen plasma etching (OPE) utilizing ignited

oxygen plasma in an evacuated state for 1 minute.

5. A second layer of photoresist 1813 was spun and heat set at the same parameters as

previously reported, part F in Figure 2.3. Followed with UV exposure in the mask aligner

with a secondary mask; exposing only 10 μm × 10 μm holes over each 50 μm × 50 μm

electrode pad, as well as, the electronic contact pads on the outer slide edge. Once

more, development and rinsing were performed with developer 352 and deionized

water with parameters as before, followed with drying with a nitrogen gun, part G in

Figure 2.3.

6. Electrode surfaces were cleaned with OPE for 1 minute. Storage was done in a

dessicator to avoid atmospheric contamination before testing. 16

The slides were completed with three arrays per slide giving a total of 54 individually isolated working electrodes per slide. The final step, in sensor construction, was an adhesion of the sterol plastic wells cut from a 96‐welled plate. The sensor construction is designed for future application and coupling with a 96‐welled plate. Herein we report experimental results from wells previously cut out of the plate and adhered to the sensor. The wells were attached with the use of a non‐conductive epoxy, shown to have no effect on the photoresist mask or the individual circuitry that comprised the array electrodes.

2.2.2 MEA Sensor Fabrication and Construction

The microelectrode arrays were based on a planar design where 10 μm x 10 μm

gold working electrodes are positioned (1 μm thick) on glass to form arrays. Adequate

adherence to glass is accomplished by thin (150 nm) layer of titanium between gold surface

and glass support base. Glass was chosen as the support base for its low electrical

conductivity. The exact geometry of these electrodes and arrays are as follows: nine 50 μm

× 50 μm base electrodes with 30 μm circuitry to connect the electrodes with an outer slide connection pad, utilized for connection to an external electrochemical analyzer, comprise half an array. Two half arrays work together to form an array with connection pads on each side of the glass slide. The three central electrodes in a nine electrode half array have short spans of 10 μm circuitry for ease in fabrication. A final layer of photoresist 1813 masks the entire slide with exception for the 10 μm × 10 μm holes allowed over each 50 μm × 50 μm pad and the connection pads on the glass slide edges. Figure 2.4 shows these half arrays both before and after the final photoresist mask is applied and treated. Figure 2.5 shows a completed array consisting of two, nine electrode, half arrays. Figure 2.6 show a finished 17 glass slide with all the individual electrodes comprising the three arrays as well as the numbering scheme developed to label each array.

Figure 2.4 Half array pictures including: final half array comprising of 9 electrodes, wiring, and electronic contact pads (A), zoomed in views of the 9 electrode test site before and after the final layer of resist is applied and holes are opened up, (B) and (C), respectively.

Figure 2.5 Compete array consisting of 18 electrodes, wiring, and electronic contact pads. Three arrays are located on each glass slide. 18

Figure 2.6 MEA and numbering scheme for completed sensor platform. Numerical designation continued as across wells; Well #2 electrode designations are 1018, Well #3 electrode designations are 1927.

Additional optical characterization information for the sensor platform, as well as, additional conformation depictions can be found in the Appendix, Figures A.1 – A.9.

2.2.3 Electrochemical Measurements and Reagents

All electrochemical testing was done using CHI 1220 Electrochemical Analyzer (CH

Instruments, TX, USA) and all experiments were done in the standard three electrode configuration. Gold electrodes of the MEA, fabricated as aforementioned, 2 mm gold electrode (termed as “macroelectrode”), or a 10 μm gold wire working electrode (CH

Instruments) were utilized as the working electrode with silver|silver chloride (Ag|AgCl) reference electrode and platinum wire counter electrode (both from CH Instruments). All potentials applied in the electrochemical testing are referred to this reference electrode.

Cyclic voltammetry (CV) and square‐wave voltammetry (SWV) were done on varying concentrations of 0.05, 0.1, 0.3, 0.5, 0.8, and 1.0 0.05 mM K3Fe(CN)6 in 0.01 M PBS (both from Sigma‐Aldrich) and recorded at the working electrode, Table 1 shows the CV and SWV 19 testing parameters. The K3Fe(CN)6 in 0.01 M PBS solutions were deoxygenated by nitrogen purge for at least 10 minutes prior to testing. Polishing of the 10 μm gold wire working electrode was done by successive polishing using 1 (bare borundum sanding paper only),

0.3, 0.1, and 0.05 alumina slurries, followed by thoroughly rinsing with distilled water. The

MEA gold working electrodes were utilized

“as is” after the oxygen plasma etch treatment described earlier. The electrochemical cell consisted of the sterol plastic well of approximately 400 μl volume, for the MEA working electrode tests, and a 25 ml glass beaker, for the 10 μm gold wire working electrode tests.

The counter and reference electrodes were inserted through the top opening, Figure 2.7, for the MEA working electrode tests, and all three electrodes were inserted through the top opening of the glass beaker for the 10 μm gold wire working electrode tests.

Figure 2.7 Electrochemical cell configuration during testing of the MEA sensor platform.

20 Table 2.1 Input parameters for cyclic and squarewave voltammetry testing. Parameter Type CV Parameter Parameter Type SWV Parameter Initial E (V) 0.5 0.5 High E (V) 0.5 Final E (V) ‐0.1 Low E (V) ‐0.1 Increment E (V) 0.004 Input Positive/Negative N Amplitude (V) 0.025 Scan Rate (V/s) 0.01 Frequency (Hz) 15.001 Segment (#) 2 Sample Interval (V) 0.001 Quiet Time (s) 2 2 Sensitivity (A/V) 1*10‐9 1*10‐9

2.3 Results and Discussion

2.3.1 Comparison of CV Results of MEA Microelectrode and 2 mm Au Electrode

Initial tests to determine if the MEA electrodes were operational and to characterize

the electrodes were done with CV measurements on the ferric/ferrocyanide redox couple.

A solution of 0.1 mM K3Fe(CN)6 in 0.01 M PBS was tested on individual MEA electrode with

potential range from 0.5 to ‐0.1 V with scan rate of 10 mV/s to prove operation. Figure 2.8

shows a typical response from the MEA electrode compared to that of a 2 mm

macroelectrode. The voltammogram gives the sigmoidal shape indicative of nonlinear

hemispherical 3‐D diffusion as expected from a microelectrode tested on the ferric/ferrocyanide couple and is in agreement with CV curves from literature.3,10,14,28

While the current is monitored, the CV testing begins at 0.6 V or 0.5 V potential for

MEA or the macroelectrode, respectively, and proceeds in a sawtooth sweeping manner towards ‐0.1, causing the iron species of K3Fe(CN)6 in solution to be reduced on the MEA electrode.

‐ K3Fe(CN)6 + e → K4Fe(CN)6

The voltammogram from the macroelectrode shows the increasing current response

typically seen in the reducing pass but there is a peak formation at approximately 0.25 V 21 due to the transient limiting diffusion in the diffusional zone formed over the electrode surface (red line in Figure 2.8). However, the MEA electrode does not have the transient diffusion limitation due to the increased nonlinear diffusion influence; thus, the voltammogram has the steady state sigmoidal appearance. Once the scanning potential reaches ‐0.1 V the scan direction reverses and the oxidative pass begins with scanning returning to initial potential, and K3Fe(CN)6 is oxidized.

‐ K4Fe(CN)6 – e → K3Fe(CN)6

Once more the macroelectrode shows the transient diffusion limited peak at approximately 0.25 V and the MEA electrode shows the steady state sigmoidal shape. The hysteresis is proportional to the width or diameter of the electrode and there is significantly less hysteresis observed in the MEA electrode voltammogram.

4.0x10-10 2.0x10-6

1.5x10-6 3.0x10-10 1.0x10-6

-10 2.0x10 -7 5.0x10

0.0 1.0x10-10 Current (A) Current (A) Current -5.0x10-7 0.0 MEA Response (Primary Axis) -1.0x10-6 2mm Response (Secondary Axis) -1.0x10-10 -1.5x10-6 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 -0.1 -0.2 Potential (V)

Figure 2.8 Comparison of cyclic voltammograms of typical MEA electrode (R10 in this case) and that of a 2 mm macroelectrode on 0.3 mM K3Fe(CN)6 in 0.01 M PBS; MEA electrode: 0.5 to 0.1 V, scan rate of 10 mV/s; macroelectrode: 0.6 to 0.1 V, scan rate of 10 mV/s. Arrows indicate the potential scanning direction.

22 2.3.2 Repeatability of CV Results for Single MEA Electrode

Figures 2.9 and 2.10 shows the results of the L9 electrode as it was tested in five successive runs with 0.1 mM K3Fe(CN)6 in 0.01 M PBS, 0.5 to ‐0.1 V with scan rate of 60

mV/s. This was done to gain a sense of the repeatability of the electrodes and to check for

sensor drift. Clearly these electrodes have a high degree of repeatability as no significant

drift is observed over the five runs tested. There was a slight difference with the initial

portion of the reducing path observed on the first cycle tested but it is negligible because

the maximum current response is monitored which lies at the end of the reducing path at ‐

0.1 V. At this maximum current response there is almost no difference between the five

cycles tested noted by the standard deviation laying atop the average, shown in Figure 2.10.

Also from the cycles there is greater observed hysteresis at the 0.5 V to 0.2 V range. It is

unclear what the cause of this influence was from, possibly the difference between using a

scanning rate of 60 mV/s compared to the typical 10 mV/s utilized on all other CV testing.

Figure 2.9 CV testing of electrode L9 in five successive runs on 0.1 mM K3Fe(CN)6 in 0.01 M PBS, 0.5 to 0.1 V with scan rate of 60 mV/s. Arrow indicates the potential scanning direction. 23

1.4x10-10

1.2x10-10

1.0x10-10

8.0x10-11

6.0x10-11

4.0x10-11 Current (A) 2.0x10-11

0.0

-2.0x10-11 MEA Average Response 1 Standard Deviation -4.0x10-11

-6.0x10-11 0.5 0.4 0.3 0.2 0.1 0.0 -0.1 Potential (V)

Figure 2.10 Average and 1 standard deviation of L9 electrode tested in 0.01 M PBS, 0.5 to 0.1 V with scan rate of 60 mV/s. Arrow indicates the potential scanning direction.

2.3.3 Repeatability of 18 MEA Electrodes in 0.3 mM and 0.5 mM K3Fe(CN)6 Solutions

All of the electrodes comprising 1 test site were tested on 0.3 mM and 0.5 mM

K3Fe(CN)6 in 0.01 M PBS with potential from 0.5 to ‐0.1 V with scan rate of 10 mV/s, in order to gain a sense of the electrode variation when each is tested with the same solution independently. Results for 0.3 mM K3Fe(CN)6 in 0.01 M PBS are shown in Figures 2.11‐2.13, and results for 0.5 mM K3Fe(CN)6 in 0.01 M PBS are shown in Figures 2.14‐2.16. Figures

2.11‐2.12 and 2.14‐2.15 show the actual voltammograms for individual 18 electrodes, and

the average and 1 standard deviation for each concentration is shown in Figures 2.13 and

2.16.

Firstly it is noted, that each individual MEA electrode has the steady state sigmoidal

shape where no transient limited peaks appear. There is a small spike in the

voltammogram of the L11 electrode at ~ 0.3 V that was caused by accidental bumping of the 24 electrochemical cell as the test was being performed. It is completely negligible because the maximum current reading is used as the response signal and this spike is far away from the

‐0.1 V potential where this reading is taken.

Secondly, there is a small amount of variation between electrodes of one test site

with both 0.3 mM and 0.5 mM K3Fe(CN)6 in 0.01 M PBS. Figure 2.17 compares the average and standard deviation of all 18 MEA electrodes at both concentrations. The responses show smaller variation at the lower 0.3 mM concentration than that of the higher 0.5 mM concentration. A possible reason for the variance may be saturation of the sensors ability to reduce and oxidize the K3Fe(CN)6 by excess analyte in solution, implicating the linear full

span output or the linear testing range of the sensor has been surpassed and the response

has become nonlinear as it approaches some asymptote current value it cannot surpass by

adding more analyte. However, later it will be shown that this is not the case as the linear

full span output is greater than 0.5 mM, and higher concentrations can be monitored in a

linear fashion. As of now, the variation increase in 0.5 mM compared to that of 0.3 mM is

believed to be a product of experimentation as these tests were performed only once for

each electrode, and duplication of the experiment with triplicate testing on the electrodes

could shed insight into the observed difference in variation.

There is a difference between responses at the tested concentrations. As expected,

the higher 0.5 mM analyte concentration gave a response of approximately 530 pA,

compared to that of approximately 350 pA for 0.3 mM analyte, as shown in Figure 2.17.

Furthermore, the average responses of the 18 MEA electrodes, with respect to both

concentrations, have non‐overlapping 1 standard deviation difference; they are significant

and different.

-10 -10 4.0x10-10 4.0x10 4.0x10 -10 -10 3.0x10-10 3.0x10 3.0x10 -10 -10 2.0x10-10 2.0x10 2.0x10 -10 -10 1.0x10-10 1.0x10 1.0x10 0.0 0.0 L11 0.0 L12

L10 Current (A) Current (A)

Current (A) Current -10 -10 -1.0x10-10 -1.0x10 -1.0x10 -10 -10 -2.0x10-10 -2.0x10 -2.0x10 0.5 0.4 0.3 0.2 0.1 0.0 -0.1 0.5 0.4 0.3 0.2 0.1 0.0 -0.1 0.5 0.4 0.3 0.2 0.1 0.0 -0.1 Potential (V) Potential (V) Potential (V)